autophagy Flashcards
Learning Objectives for Autophagy
To introduce the process of autophagy and why it is important.
Learning Objectives
● Understand the basic concept of autophagy
● Understand the need for regulation
● Understand the physiological roles of autophagy
● Understand how yeast genetics can be used as a model system to study
autophagy
To provide an overview of autophagy in disease
Learning Objectives
● Understand the involvement of autophagy in cancer.
● Understand the involvement of autophagy in neurodegenerative diseases such as Huntington’s and Parkinson’s disease.
● Understand the potential and limitations of autophagy-based therapies.
For simple understanding, what is autophagy?
A process to digest intracellular material.
- vesicles are made in the cytosol that capture components of the cytosol such as organelles or proteins. Ultimately these are isolated from the rest of the cytosol, fusion with lysosomes occurs and the components are degraded
What are the 6 steps of the process of autophagy?
1: Initiation - Autophagy is initiated in response to damaged organelles or cellular stressors such as nutrient deprivation or oxidative stress
2: Nucleation: Phagophore (isolation membrane) begins to form which involves recruitment of Beclin1 protein. The phagophore expands and engulfs portions of the cytoplasm, such as damaged organelles or protein aggregates
3: Elongation: Phagophore elongates and closes forming a a double-membrane vesicle known as autophagosome
4: Maturation - Autophagosome undergoes maturation which enables it to fuse with lysosomes. this forms an autolysosome
5: Degradation: within the autolysosome the sequestered material is exposed to lysosomal enzymes leading to the breakdown of the sequestered material. The resulting breakdown products are recycled back into the cell
- Termination - the process ends with the recycling of the autolysosome and release of recycled material back into the cytoplasm
What physiological processes is autophagy needed for?
- removing damaged components e.g. from daily wear and tear
- Signalling
- Recycling nutrients
- Differentiation of cells/cellular remodelling
-Killing intracellular pathogens
List the key characteristics of the 3 protein degradation pathways - proteasome pathway, autophagy pathway, chaperone mediated autopghagy
Macroautophagy
- Lysosomal
- bulk digestion
- can remove whole organelles
- molecules released support metabolism
proteosome pathway:
- non lysosomal
- degrades individual proteins
- Major turnover route for short lived proteins
chaperone mediated autophagy/microautophagy
- lysosomal
- degrades individual proteins
- turns over specific long lived proteins
What is the ONLY mechanism of degrading organelles?
Autophagy
What cell type is autophagy needed to form?
Erythrocytes
Yeast experiment for autophagy
- If you took yeast strains with deficient proteases that could not digest things in the vacuole efficiently and you starve them, you start to see accumulation of material
- It was speculated these were autophagosomes catching material in the cytosol, fusing with the vacuole but they can’t be digested hence the accumulation
What do cells lacking autophagy accumulate?
Protein aggregates
What causes the common phenotype of neurodegenerative diseases?
Accumulation of protein aggregates
What is Huntington’s disease caused by?
What does effect does this have on the protein?
PolyQ in Huntingtin protein
PolyQ causes Huntingtin protein to misfold - the longer the polyQ region the more likely the protein will misfold
Explain the mechanism of Huntington’s disease
4 points
- With polyQ Huntingtin, misfolding and aggregation can occur (this only becomes a problem when rate of misfolding Huntingtin proteins is higher than the capacity to degrade them)
-With age autophagy capacity decreases hence why Huntington’s is more prevalent with age
- Usually when aggregation of Huntingtin proteins occurs, they are ubiquitinated and some undergo proteasomal degradation and are digested
- If there are too many aggregates for proteosomal degradation, the aggregates are clumped together into one big aggresome which would normally targeted by autophagy for degradation
List and explain mechanisms of toxicity in relation to Huntington’s disease
5 points
- One way could be that due to the misfolding, the protein loses its original function
- Could be the toxic oligomers - perhaps they damage or override the protein
- Could be the aggresomes
- Proteins that normally binds huntingtin could also be included in the aggregates and then degraded by autophagy
-this ultimately leads to loss of neuronal activity
Simply, what causes parkinsons?
What type of neurones does parkinson normally affect?
Compare cases of Parkinson’s and Huntington’s in terms of what % of cases are familial
- Aggregated proteins in cytosol of cells - mainly alpha-syn aggregates
- Mitochondria accumulation
- Specifically affects dopaminergic neurones
In Huntington, most cases are familial. In Parkinson’s, only 5-10% of cases are familial
What happens in chaperone mediated autophagy, generally speaking?
Individual proteins go through LAMP2 receptor on lysosomes and enter the lysosome for degradation
What protein is the main cause of Parkinson’s? How common is it that a mutation in this causes Parkinson’s?
Aggregates of alpha-syn
rare - 5-10% of cases
How does mutated alpha-syn contribute to the development of the Parkinson’s disease?
- Alpha syn is normally degraded by chaperone mediated autophagy. in mutated form, alpha syn blocks the pathway
-Alpha-syn mutation blocks the chaperone mediated pathway which in turn also blocks other proteins being taken up via the pathway which causes dysregulation of cells
What is the effect of alpha-syn on what pathway? How can this cause a general dysregulation of cells
Alpha-syn blocks the chaperone mediated pathway
-alpha-syn engages the receptor but it cannot be transferred across the membrane
- through alpha-syn blockage of the chaperone mediated pathway, other proteins are also blocked from being taken up via this pathway which can cause dysregulation of cells
The accumulation of what 2 things leads to Parkinson’s?
-Accumulation of alpha-syn protein
- accumulation of damaged mitochondira
Why is accumulation of damaged mitochondria a bad thing?
- Mitochondria are the main source of ROS in the body
- Damaged mitochondria release higher levels of ROS
- ROS damages cellular compartments
What are the 2 most prominent genes in familial Parkinson’s?
What are they?
How often are they found in what cases?
PINK1
- Is a mitochondrial kinase
- mutation only found in 5-10% of sporadic early onset parkinsons
PARKIN
- is a ubiquitin ligase - responsible for tagging damaged mitochondria with ubiquitin which marks them for degradation through autophagy
- mutation found in 10-15% of sporadic early onset parkinson
Explain how PINK1 and PARKIN play a role in Parkinson’s
3 points
- Normally when a mitochondria becomes damaged - PINK1 and PARKIN work together which leads to ubiquitination of that mitochondria and its ability to be sequestered within an autophagosome
-If you lack PINK1 or PARKIN, the mtch cannot be targeted. This means mtch accumulate over time which causes release of more ROS, more oxidative damage which results in more proteins being misfolded and more organelles being damaged
-This is a feedback system hence why symptoms worsen with time
in leymans terms, what causes cancer?
What is the cause of most cancers
- cancer is caused by accumulation of DNA damage
- Most cancers begin with a mutation in a gene required for normal function e.g. growth, cell death, DNA repair
Is autophagy tumour repressive?
Yes
What mutation do patients with cancers often have?
What is the normal function of the gene when it isn’t mutated?
Mice example
- Beclin1 mutation often seen in cancer
-Beclin1 is a gene required for autophagosome formation
Less Beclin1 = can accumulate more DNA damage
- Can knockout Beclin1 in mice = mouse die from tumours within 18 months
What process do cells use inside growing tumours to survive?
why do they do this?
cells inside growing tumours use autophagy to survive - because the environment inside growing tumours is low in nutrients and oxygen
Which common cell regulation process does autophagy inhibit, and vice versa?
Autophagy inhibits apoptosis
What is the leyman purpose of autophagy vs apoptosis in cells?
Apoptosis keeps cells alive
apoptosis destroys cells
What is Beclin1? What is its general function?
- Mutation in this Beclin1 gene is often seen in cancer patients
- Beclin1 is part of a complex with a lipid kinase that makes lipids which leads to autophagosome formation
Is Beclin1 lost in tumours?
Yes
Where are Bcl proteins normally found?
Describe the role of Bcl2 proteins in apoptosis
3 points
Bcl2 is usually found on the outer membrane of mitochondria
Bcl2 controls how much cytochrome is released from mitochondria, this is important for driving apoptosis
- Bcl2 suppresses release of cytochrome from mitochondria
Bcl2 is an inhibitor of apoptosis
What happens when Beclin1 and Bcl2 interact?
- Bcl2 is taken away from the mitochondria, which means the mitochondria are free to cause apoptosis
- Beclin1 is taken away from its complex with lipid kinase, so autophagosome formation no longer occurs
Why is ‘losing’ Beclin1 a bad thing when it comes to tumours?
if you lose Beclin1, bcl2 is free to bind to mitochondria which inhibits apoptosis - not what you want to happen inside tumour cells
Beclin1 and Bcl2 are known to interact with each other. What happens when you activate autophagy?
When you activate autophagy, Beclin1 is phosphorylated and returns to its lipid kinase complex, therefore autophagosome formation needed for autophagy can occur. At the same time, Bcl2 returns to the mitochondria, inhibiting apoptosis
-When you activate autophagy you automatically inhibit apoptosis and the cell is driven towards a survival state
Autophagy is anti-oncogenic and pro-oncogenic.
What does oncogenic mean?
List how is it anti-oncogenic and pro-oncogenic
Oncogenic = a mutated gene that has potential to cause cancer
anti-oncogenic:
- Cell homeostasis
- Damage removal
- Reduced ROS
- Reduced inflammation
pro-oncogenic
- Cells can survive in low oxygen/nutrient environments
- Prevention of apoptosis
- Survival during chemotherapy
Explain 3 examples of autophagy therapeutics in cancer
- If you have tumours, inhibit autophagy to prevent cells from surviving metabolic stress inside the tumour as the tumour grows
- Inhibit autophagy to increase apoptosis e.g. during chemotherapy
-Elevate autophagy in healthy individuals to remove damaged cells and reduce likelihood of cancer occurring in the first place
